IL-33 Regulates the Iga-Microbiota Axis to Restrain IL-1Α– Dependent Colitis and Tumorigenesis

IL-33 regulates the IgA-microbiota axis to restrain IL-1α– dependent colitis and tumorigenesis

Ankit Malik, … , Peter Vogel, Thirumala-Devi Kanneganti

J Clin Invest. 2016;126(12):4469-4481. https://doi.org/10.1172/JCI88625.

Research Article Immunology

Inflammatory bowel diseases (IBD) affect over 5 million individuals in the industrialized world, with an increasing incidence rate worldwide. IBD also predisposes affected individuals to development of colorectal cancer, which is a leading cause of cancer-related deaths in adults. Mutations in genes encoding molecules in the IL-33 signaling pathway are associated with colitis and colitis-associated cancer (CAC), but how IL-33 modulates gut homeostasis is unclear. Here, we have shown that Il33-deficient mice are highly susceptible to colitis and CAC. Mechanistically, we observed that IL-33 promoted IgA production from B cells, which is important for maintaining microbial homeostasis in the intestine. Il33- deficient mice developed a dysbiotic microbiota that was characterized by increased levels of mucolytic and colitogenic bacteria. In response to chemically induced colitis, this microbial landscape promoted the release of IL-1α, which acted as a critical driver of colitis and CAC. Consequently, reconstitution of symbiotic microbiota or IL-1α ablation markedly ameliorated colitis susceptibility in Il33-deficient animals. Our results demonstrate that IL-33 promotes IgA production to maintain gut microbial homoeostasis and restrain IL-1α–dependent colitis and CAC. This study therefore highlights modulation of IL-33, IgA, IL-1α, and the microbiota as a potential therapeutic approach in the treatment of IBD and CAC.

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IL-33 regulates the IgA-microbiota axis to restrain IL-1α–dependent colitis and tumorigenesis

Ankit Malik,1 Deepika Sharma,1 Qifan Zhu,1,2 Rajendra Karki,1 Clifford S. Guy,1 Peter Vogel,3 and Thirumala-Devi Kanneganti1 1Department of Immunology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA. 2Integrated Biomedical Sciences Program, University of Tennessee Health Science Center, Memphis, Tennessee, USA. 3Animal Resources Center and the Veterinary Pathology Core, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.

Inflammatory bowel diseases (IBD) affect over 5 million individuals in the industrialized world, with an increasing incidence rate worldwide. IBD also predisposes affected individuals to development of colorectal cancer, which is a leading cause of cancer-related deaths in adults. Mutations in genes encoding molecules in the IL-33 signaling pathway are associated with colitis and colitis-associated cancer (CAC), but how IL-33 modulates gut homeostasis is unclear. Here, we have shown that Il33-deficient mice are highly susceptible to colitis and CAC. Mechanistically, we observed that IL-33 promoted IgA production from B cells, which is important for maintaining microbial homeostasis in the intestine. Il33-deficient mice developed a dysbiotic microbiota that was characterized by increased levels of mucolytic and colitogenic bacteria. In response to chemically induced colitis, this microbial landscape promoted the release of IL-1α, which acted as a critical driver of colitis and CAC. Consequently, reconstitution of symbiotic microbiota or IL-1α ablation markedly ameliorated colitis susceptibility in Il33- deficient animals. Our results demonstrate that IL-33 promotes IgA production to maintain gut microbial homoeostasis and restrain IL-1α–dependent colitis and CAC. This study therefore highlights modulation of IL-33, IgA, IL-1α, and the microbiota as a potential therapeutic approach in the treatment of IBD and CAC.

Introduction infection (11–15). In addition, IL-33 has been reported to pro- Inflammatory bowel diseases (IBD), such as Crohn’s disease mote antiviral responses by enhancing cytokine production (CD) and ulcerative colitis (UC), affect approximately 1.6 mil- from cytotoxic T cells (16) and to exacerbate arthritis by hyper- lion individuals in the USA and 3.5 million individuals in Europe, activating mast cells (17). constituting a major health problem (1, 2). IBD involves chronic Multiple studies have demonstrated that IL-33, ST2 (the inflammation in the intestine with episodes of acute flare-ups binding site for IL-33), and soluble ST2 (sST2, a decoy receptor) that manifest as abdominal pain, diarrhea, rectal bleeding, and are upregulated in the mucosa of patients with IBD, and polymor- body weight loss (1, 2). The mucus and the epithelial layer form phisms in IL33 and ST2 have been associated with the develop- a physical barrier between the microbiota and underlying host ment of IBD (18–22). Expression levels of ST2 correlate inversely immune system, and deterioration of this barrier is prominent with the grade of colon tumors, suggesting that IL-33 signaling in IBD patients (3, 4). In addition, rodent studies have linked is protective in CAC (22). However, the role of IL-33 in IBD and disruption of the epithelial barrier to cytokine imbalances in colon tumorigenesis is unclear. Genetic ablation of Il33 in mice the gut that eventually lead to the development of IBD (5). IBD of a mixed genetic background lowered clinical symptoms in the also predispose patients to the development of colorectal can- early phase of experimental colitis, but also delayed the resolu- cer, referred to as colitis-associated cancer (CAC) (6). Colorec- tion of inflammation (23). Administration of recombinant IL-33 tal cancer is a leading cause of adult cancer-related deaths, with was also shown to ameliorate colitis in Il33-sufficient (WT) mice, 160,000 cases being diagnosed annually in the USA, and colitis suggesting a protective role for IL-33 in colitis (24, 25). By uti- is associated with approximately 90% of these cases (7). lizing Il33–/– mice generated on a congenic C57BL/6 background IL-33 is a member of the IL-1 cytokine family that is and employing cohousing strategies and littermate controls, we expressed constitutively in epithelial cells and reticular fibro- show that IL-33 protected from colitis and CAC. IL-33 was criti- blasts and can be induced in immune cells (8). IL-33 is usually cal to maintaining gut homeostasis by regulating IgA production localized in the nucleus and released upon cell death (9). How- in the colon. Reduced intestinal IgA in Il33–/– mice led to a dys- ever, unlike IL-1β and IL-18, which require cleavage by caspase biotic microbiota characterized by increased levels of mucolytic 1 for bioactivity, IL-33 is bioactive in the full-length form (9, and colitogenic bacteria. Consequently, administration of dex- 10). IL-33 serves as an amplifier of the type 2 cytokine response tran sulfate sodium (DSS) in Il33–/– mice led to increased release

from TH2, ILC2, mast cells, and alternatively activated macro- of proinflammatory IL-1α, which was a critical driver of CAC. phages during asthma, allergic hypersensitivity, and helminth Remarkably, cohousing Il33–/– mice with WT controls equilibrated the level of mucolytic bacteria and thereby protected ll33–/– mice from colitis and epithelial hyperplasia. These findings Conflict of interest: The authors have declared that no conflict of interest exists. Submitted: May 18, 2016; Accepted: September 15, 2016. reveal a critical role for IL-33 in regulating the immune responses Reference information: J Clin Invest. 2016;126(12):4469–4481. doi:10.1172/JCI88625. and microbiota in the gut.

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Figure 1. IL-33 decreases susceptibility to DSS-induced colitis. (A) Body weight change, (B) disease activity index, and (C) relative survival of WT and Il33–/– mice during administration of 3% DSS in drinking water. (D) Body weight change and (E) disease activity index of mice during administration of 2% DSS in drinking water. (F) Colon length measurement and (G) representative pictures of colons from WT and Il33–/– mice at the indicated days after 2% DSS administration. (H) H&E staining. Original magnification, ×10. (I) Histological analysis of colon tissue from mice at the indicated days after 2% DSS administration. (J) Incidence and levels of bacteria in mesenteric lymph nodes (MLN) of mice at day 4 and day 8 after 2% DSS administration. Data represent 2 independent experiments and are presented as mean ± SEM (A–I) or median (J). Each symbol represents an individual mouse. n = 8–10 mice per time point per group. Data were analyzed by 2-way ANOVA (A, B, D, and E), log-rank (Mantel-Cox) test (C), or Kruskal-Wallis test (F and J) followed by Holm-Šídák post test. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Results body weight loss and disease activity score when compared IL-33 regulates the development of colitis and associated cancer. with WT mice (Figure 1, A and B). Further, 100% of the Il33–/– To determine the role of IL-33 in pathogenesis of IBD, WT and mice reached humane end point of the experiment (>20% body Il33–/– mice were administered with DSS in drinking water for 6 weight loss) by day 8, while all the WT mice survived (Figure days, followed by regular drinking water. Following administra- 1C). At a lower dose of DSS (2%), Il33–/– mice still displayed sig- tion of 3% DSS, Il33–/– animals displayed significantly greater nificantly greater weight loss and disease activity scores (Figure

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Figure 2. IL-33 decreases susceptibility to CAC. (A) Body weight loss of WT and Il33–/– mice injected with AOM on day 0 and administered 3 rounds of 2% DSS in drinking water. (B and D) Quantification of the number of tumors in the colon at day 82 after AOM injection. (C) Representative images of distal and middle colon at day 82 after AOM administration. (E) Histological analysis and (F) H&E staining of colon sections at day 82 after AOM injection. Original magnification, ×2. Data represent 2 independent experiments and were analyzed by 2-way ANOVA followed by Holm-Šídák post test (A) or Mann-Whitney U test (B). Error bars represent mean ± SEM. (A, B, and D) and each symbol represents an individual mouse with 5 mice per group. **P < 0.01; ***P < 0.001; ****P < 0.0001.

mice were injected with DNA damaging agent azoxymethane (AOM), followed by 3 rounds of low-dose (2%) DSS treatment (27). Il33–/– mice lost significantly more body weight during each round of DSS treatment (Figure 2A). At the end of the experiment, WT mice exhibited few small tumors restricted to the distal colon (Figure 2, B–D). In contrast, Il33–/– mice exhibited a significantly increased number of tumors that were of a larger size and dis- tributed throughout the distal and middle colon (Figure 2, B–D). Histological analysis revealed few low-grade adenomas in the colons of WT mice, whereas both low- and high-grade adenomas were observed more frequently in the colons of Il33–/– mice (Figure 2, E and F). These data demonstrate that IL-33 protects mice from colitis and CAC. Lack of IL-33 leads to increased secretion of IL-1α during DSS administration. To determine the immunological basis of increased colitis and CAC in Il33–/– mice, cytokine and chemokine production were analyzed in the colon during DSS administration. Day 4 was considered as a preclinical time point because there was no difference in body weight loss between WT and Il33–/– mice at that time, while day 8 was considered an acute time point because significant differences in weight change and disease activity score were observed (Figure 1D). In the colon explants, significantly increased levels of IL-1α were observed at both the preclinical time point (day 4) and acute time point (day 8) during DSS administration (Figure 3A). In contrast with the early increase in IL-1α, there was no difference in levels of the neutrophil chemokine KC (also known as CXCL1), granulocyte-CSF (G-CSF), IL-6, IL-1β, macrophage inflammatory protein-1α (MIP-1α, also known as CCL3), IL-10, and IFN-γ–induced protein 10 (IP-10, also known as CXCL10) at day 4, while they were significantly enhanced at day 8 (Figure 3A and Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI88625DS1). Lev- els of other cytokines, such as IL-17, granulocyte-macrophage CSF 1, D and E). Shortening of colon length is a feature of acute coli- (GM-CSF), monocyte chemoattractant protein-1 (MCP-1), TNF, tis (26), and Il33–/– mice had significantly shorter colons at day and CCL5, were unchanged during the course of the experiment 8 (Figure 1, F and G). Histological analysis revealed progressive (Supplemental Figure 1). These data suggest that the release of increase in inflammation, ulceration, edema, and hyperplastic IL-1α precedes the enhanced inflammatory response in Il33–/– mice proliferation of epithelial cells in the colons of Il33–/– mice dur- in response to DSS administration. While IL-1α was upregulated ing DSS administration (Figure 1, H and I). Breakdown of the at protein and transcript levels by day 8, the levels of IL-1α protein epithelial barrier in Il33–/– mice was associated with dissemina- and transcript were similar between WT and Il33–/– mice at day 4 tion of bacteria to the draining lymph nodes by day 8 (Figure during DSS administration (Figure 3, B and C). Therefore, DSS 1J). Together, these data demonstrate that IL-33 ameliorates administration leads to the release of the preformed pool of IL-1α susceptibility to DSS-induced colitis. early during disease progression. IBD is a strong risk factor for development of CAC (6). To To determine the cellular source of IL-33 and IL-1α in the determine the role of IL-33 in development of chronic colitis colon, we assessed for expression of Il33 and Il1a in epithelial and CAC, we utilized an established model of CAC wherein (Epcam+CD45–) and immune cells (CD45+Epcam–) from the

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Figure 3. Lack of IL-33 leads to early release of pathogenic IL-1α. See also Supplemental Figure 2. (A) Quantification of cytokines in supernatants of colon explants and (B) quantification of IL-1α in clarified homogenates of colons of WT and Il33–/– mice at indicated days after DSS administration. (C) qRT-PCR analysis of Il1a expression in whole colon tissue at indicated days after DSS administration. n = 8–10 mice per time point per group. (D) Body weight loss and (E) disease activity index of WT and Il33–/– mice during DSS and control IgG or anti–IL-1α antibody administration. (F) Colon length measurement and (G) representative colon images at day 8 after DSS. (H) Representative images of H&E-stained colon sections and (I) colon histology analysis at day 8 after DSS. Original magnification, ×10. n = 5 mice for WT+CIgG and 9 to 10 mice for other groups. Data represent 2 independent experiments and were analyzed by Kruskal-Wallis test (A, B, C, F, and I) or 2-way ANOVA (D and E) followed by Holm-Šídák post test. Error bars represent mean ± SEM, and each symbol represents an individual mouse. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

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Figure 4. Genetic ablation of Il1a prevents colitis and CAC in Il33–/– mice. (A) Body weight loss and (B) disease activity index of WT, Il33–/–, and Il33–/–Il1a–/– mice during AOM/DSS administration. (C) Colon length measurement and (D) representative colon images at day 8 after DSS. (E and G) Colon histology analysis at day 14 after AOM and (F) representative images of H&E-stained colon sections. Original magnification ×10. n = 5 (WT); n = 9 (Il33–/–); n = 7 for (Il33–/– Il1a–/– ). (H) Body weight loss of WT and Il33–/– mice injected with AOM on day 0 and administered 3 rounds of 2% DSS in drinking water. (I) Quantification of the number of tumors in the colon at day 49 after AOM injection (J) Representa- tive images of distal and proximal colon at day 49 after AOM administration. Original magnification, ×10. n = 10 (WT); n = 7 (Il33–/–); n = 10 (Il33–/–Il1a–/–). Data represent 2 independent experiments and were analyzed by Kruskal-Wallis test (C, E, and I) or 2-way ANOVA (A, B, and H) followed by Holm-Šídák post test. Error bars represent mean ± SEM, and each symbol represents an individual mouse. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

epithelia and antigen-presenting cells (CD45+CD90–MHCII+) antigen-presenting cells, but was not detectable in the CD90+ and CD90+ lymphocytes (CD45+CD90+MHCII–) from the lami- lymphocytes (Supplemental Figure 2C). Expression of Il1a was na propria. IL-33 was detectable in the colon tissue of WT mice upregulated by epithelial cells, antigen-presenting cells, and under homeostatic conditions and was increased at both pro- the CD90+ lymphocytes after DSS administration (Supplemen- tein and RNA levels at day 8 (Supplemental Figure 2, A and B tal Figure 2C). Therefore, both epithelial and immune cells and Supplemental Methods), consistent with previous reports of the colon contributed to increased production of IL-33 and (24, 25). Increased Il33 expression was observed in both epithe- IL-1α during DSS treatment. lial cells and immune cells of the epithelial fraction. Within the To determine the mechanism of early IL-1α release during lamina propria fraction, expression of Il33 was increased in the DSS treatment, we evaluated inflammasome activation by analyz-

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Figure 5. IL-33 promotes intestinal IgA production. (A and B) Immunoglobulin ELISA from colon explants and sera from naive WT and Il33–/– mice. (C) Immunoglobulin ELISA from colon explants at days 0, 4, and 8 after AOM administration. (D) Quantification of CD138+IgA+ cells (intracellular stain for IgA) in the colon lamina propria of naive WT and Il33–/– mice by flow cytometry. (E) Representative images for immunohistochemistry for IgA in colon sections from naive WT and Il33–/– mice. Original magnification, ×40. (F) Immunoglobulin ELISA from colon explants from naive WT, Il33–/–, Il33–/–Il1a–/–, or Il1a–/– mice. (G) Immunoglobulin ELISA from media supernatants or (H) percentage and (I) number of IgA+ B cells from WT splenic B cells (CD19+) treated with LPS, TGF-β, and/or IL-33 for 4 days. (J) qRT-PCR analysis of Pigr expression in whole colon tissue at indicated days after DSS administration. Data represent 3 independent experiments (A–D, F, and J) or 3 technical replicates that are representative of 3 independent experiments. Data in G–I were analyzed by Kruskal-Wallis test followed by Holm-Šídák post test. Error bars represent mean ± SEM, and each symbol represents an individual mouse. **P < 0.01; ***P < 0.001; ****P < 0.0001.

ing caspase 1 maturation in the colon homogenates. There was no IL-1α is a critical driver of colitis and CAC. We posited that difference in activation of caspase 1 (observed by the presence of the enhanced IL-1α release is a critical instigating event in the the p10 band) in the colons of WT and Il33–/– mice at day 4, dem- induction of colitis in Il33–/– mice. To test this hypothesis, IL-1α onstrating that IL-33 does not regulate inflammasome activation was depleted in the Il33–/– mice during early time points of DSS to mediate early IL-1α release (Supplemental Figure 2D). How- administration by injecting IL-1α–neutralizing antibody. Anti– ever, increased ulceration and necrotic cellular morphology were IL-1α treatment significantly ameliorated body weight loss and observed at day 4 and day 8 in the colon sections of Il33–/– mice other clinical signs of the disease in Il33–/– mice (Figure 3, D and after DSS administration (Figure 1, H and I, and Supplemental Fig- E). Anti–IL-1α treatment also significantly protected Il33–/– mice ure 2E), suggesting that enhanced cell death leads to early IL-1α from colon shortening and histological changes associated with release from the Il33–/– colonocytes. DSS administration (Figure 3, F–I).

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Similar to results with antibody-mediated neutralization of cytokines associated with ILC2 and TH2 response, IL-5, IL-13, and IL-1α, mice deficient in both Il33 and Il1a (Il33–/–Il1a–/–) were pro- amphiregulin, were similar in the colons of WT and Il33–/– mice at tected from AOM and DSS–induced body weight loss, clinical day 0 and day 4 and increased in the colons of Il33–/– mice at day 8 signs of disease, colon shortening, and histological changes when (Supplemental Figure 5A). We also evaluated the number of ILC2s –/– – + – + + + – compared with Il33 mice (Figure 4, A–E). Epithelial hyperpla- (CD19 CD45 Lin CD90.2 CD127 GATA-3 ) and TH2 cells (CD19 sia is an early event in tumorigenesis, which normally correlates CD45+Lin+CD90.2+CD127+GATA-3+) in the colons of WT and Il33–/– with the extent of inflammation after AOM and DSS treatment. mice during DSS administration. We found that the number of ILC2 –/– We therefore evaluated the colonic epithelial hyperplasia in WT, and TH2 cells in the colons of Il33 mice were similar to WT levels Il33–/–, and Il33–/–Il1a–/– mice at day 14 of AOM and DSS treatment. at day 0. At day 8, the number of ILC2s remained similar, but the –/– –/– Consistent with decreased inflammation in the colons of Il33 number of TH2 cells was increased in the colons of Il33 mice (Sup- –/– –/– –/– Il1a mice, a lower proportion of Il33 Il1a mice showed epithe- plemental Figure 5, B–D). Increase in the number of TH2 cells cor- lial hyperplasia (30%) when compared with Il33–/– mice (100%) related with increased levels of Il5 and Il13 and the extent of inflam- (Figure 4, E–G). Therefore, genetic ablation of Il1a also protects mation in the Il33–/– mice. These data are consistent with findings Il33–/– mice from epithelial hyperplasia. To specifically deter- from Waddell et. al., who showed that the colonic type 2 cytokine mine whether tumor development in Il33–/– mice is decreased by response is preserved in Il33–/– mice after oxazolone treatment (29). Il1a ablation, we treated WT, Il33–/–, and Il33–/–Il1a–/– mice with Expression of epithelium-healing genes, such as Il22 and its AOM followed by 3 rounds of DSS. Il33–/–Il1a–/– mice lost signifi- associated antimicrobial peptides (AMPs) Reg3g and Reg3b, was cantly lower body weight when compared with Il33–/– mice during also unperturbed at day 4 of DSS administration (Supplemen- each round of DSS administration (Figure 4H). Consistent with tal Figure 6A). Furthermore, expression of other AMPs such as decreased hyperplasia at day 14, Il33–/–Il1a–/– mice also harbored lipocalin, pentraxin, and S100a9 (Supplemental Figure 6B) and a significantly reduced number of tumors in their colons at day epithelial tight junction proteins occludin and ZO1 (Supplemen- 49 when compared with Il33–/– mice (Figure 4, I and J). The num- tal Figure 6C) were also similar between the colons of WT and ber of tumors in Il33–/–Il1a–/– mice was in fact similar to that in WT Il33–/– mice until day 8 (Supplemental Figure 6, A–C). Schiering et mice. Therefore, epithelial hyperplasia data at day 14 and tumor al. have also shown that IL-33 promotes differentiation of Tregs burden at day 49 clearly demonstrate that genetic ablation of Il1a and thereby protects lymphopenic mice from colitis resulting protects Il33–/– mice from CAC. from infusion of naive T cells (30). However, there was no defect Il1a–/– mice themselves were protected from acute colitis after in the number of Tregs in the colons of Il33–/– mice at day 0, and 4% DSS administration (Supplemental Figure 3, A and B), consis- they were increased during DSS administration (Supplemental tent with a previous report (28). Moreover, upon AOM and DSS Figure 6D). Collectively, these data demonstrate that perturba- treatment with a 3.5% DSS dose, Il1a–/– mice lost significantly less tion in production of mucins, type 2 cytokines, AMPs, tight junc- body weight than WT controls at chronic time points of days 66 tion proteins, and epithelial restitution factors is not observed in to 90 (Supplemental Figure 3C). Histological analysis revealed the colons of Il33–/– mice until overt inflammation is established. reduced inflammation and epithelial dysplasia in the Il1a–/– colon Therefore, while perturbation in these pathways may promote (Supplemental Figure 3, D–G). Therefore, IL-1α is an important disease pathogenesis, they are unlikely to be responsible for the driver of colitis and CAC. early release of pathogenic IL-1α. IL-33 promotes intestinal IgA production. Next, we examined Intestinal IgA is a critical factor in promoting gut homoeo- the mechanism behind increased IL-1α release from Il33–/– colono- stasis. Intestinal IgA has been shown to neutralize toxins and cytes after DSS administration. In order to reach the colonocytes, preferentially target IBD-promoting gut bacteria for clearance DSS must first penetrate the mucus barrier. IL-33 has been shown by M cells and lamina propria macrophages (31). Further, IgA to upregulate the production of mucus after helminth challenge deficiency predisposes humans to gut infections and IBD and and oxazolone treatment (14, 29). We therefore examined pos- increases severity of DSS-induced colitis in mice (32–34). There- sible defects in the mucus production in Il33–/– mice. There were fore, we evaluated the level of intestinal IgA in Il33–/– mice. Il33–/– no significant differences in the expression of mucins Muc1, Muc2, mice had significantly decreased levels of IgA in colon explants, Muc3, Muc4, or Muc5ac at basal or preclinical time points during while the levels of other immunoglobulins — IgM, IgG2b, IgG2c, DSS administration between WT and Il33–/– colons (Supplemental IgG3, and IgG1 — were similar to WT levels (Figure 5A). Levels Figure 4A). Further, the number of goblet cells was similar between of IgA and other immunoglobulins were similar in sera, suggest- the colons of WT and Il33–/– mice under homeostatic conditions ing a gut-specific role of IL-33 in regulating IgA production (Fig- (day 0). Also, consistent with increased colitis in Il33–/– mice, fre- ure 5B). Furthermore, levels of intestinal IgA stayed low in the quency of goblet cells was decreased in the colons of Il33–/– mice Il33–/– mice during DSS administration (Figure 5C). Decrease in during DSS treatment (Supplemental Figure 4, B and C). IL-33 has intestinal IgA was also confirmed by a decrease in the number of also been shown to be an amplifier of type 2 cytokine response IgA-producing plasma cells (gated as CD138+IgA+ with intracel- –/– from TH2 and ILC2 cells after helminth and allergen challenge lular staining for IgA) in the colons of Il33 mice (Figure 5, D and (14, 29). Monticelli et. al. showed that exogenous IL-33 adminis- E). The intestinal IgA level in Il1a–/– mice was similar to the WT tration expands ILC2s in the GALT and upregulates amphiregulin level, while the level in Il33–/–Il1a–/– mice was similar to the level production (24). However, the impact of IL-33 deficiency on ILC2 in Il33–/– (Figure 5F). Therefore, protection from colitis and CAC

and TH2 response in the gut under homoeostatic conditions or dur- that was conferred by IL-1α blockade or deficiency was indepen- ing DSS administration has not been directly tested. The levels of dent of modulation of intestinal IgA production.

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Figure 6. IL-33 regulates gut microbial homeostasis. (A) qPCR analysis of indicated bacteria in the colon contents from naive WT and Il33–/– mice. (B) Fluo- rescent in situ hybridization with Akkermansia-specific probe in colon sections from naive mice. Dashed white line represents the epithelium. Scale bars: 8 μm. (C) qPCR analysis of indicated bacteria from stool samples of naive WT and Il33–/– mice treated with water or metronidazole (Mtz) for 5 days. (D) Quantification of IgA in fecal pellets after 5 days of metronidazole treatment. (E) IL-1α in supernatants of colon explant cultures at day 4 after DSS administration. (F) Body weight loss and (G) disease activity index of mice during DSS or metronidazole and DSS administration. (H) Colon length measurement and (I) representative colon images at day 8 after DSS or metronidazole and DSS administration. (J) Representative images of H&E-stained colon sections and (K) colon histology analysis at day 8 after DSS. Original magnification, ×10. Data represent 2 independent experiments and were analyzed by 2-way ANOVA (F and G) or Kruskal-Wallis test (C–E, I, and K), followed by Holm-Šídák post test. Error bars represent mean ± SEM, and each symbol represents an individual mouse. n = 5 (WT); n = 7 for (Il33–/–). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

To assess whether IL-33 promotes IgA production from B body weight loss, colon shortening, and histological changes asso- cells, we utilized an in vitro class-switching assay. WT splenic B ciated with DSS administration (Figure 6, F–K). In fact, metronida- cells were stimulated with LPS in the presence of IL-33, TGF-β, zole-treated Il33–/– mice were indistinguishable from WT controls or both. TGF-β significantly increased the production of IgA and after DSS administration. decreased production of IgM and IgG3 (Figure 5G), consistent Metronidazole treatment also led to a significant decrease in with previous reports (35). Addition of IL-33 alone did not affect the relative abundance of Lactobacillus and Clostridia, bacterial the isotype or quantity of immunoglobulin production. However, groups that have been shown to be protective in colitis (46, 47), and addition of both TGF-β and IL-33 further enhanced IgA produc- did not significantly affect the level of SFB (Figure 6C). Therefore, tion (Figure 5G). Increase in IgA secretion was consistent with colitis susceptibility in Il33–/– mice is independent of these bacte- increase in both the proportion and total number of IgA+ cells in B rial groups. Metronidazole administration also decreased the level cells treated with both TGF-β and IL-33 (Figure 5, H and I). IL-33, of Prevotella (Figure 6C), which has been shown to be pathogenic however, did not affect the expression of polymeric immunoglob- in this model of colitis (48). We therefore employed a strategy to ulin receptor (Pigr), a receptor required for secretion of IgA into determine the severity of DSS colitis after increasing the level of the lumen (Figure 5J). Therefore, IL-33 synergizes with TGF-β to Akkermansia while simultaneously depleting Prevotella. To this promote IgA production, and absence of IL-33 leads to decreased end, mice were treated with a broad-spectrum antibiotic cocktail IgA levels in the intestine. that we have shown previously to boost the level of Akkermansia, Colitis susceptibility in Il33-deficient mice is dependent on dys- but decrease the level of Prevotella in the gut (49). Pretreatment of biotic microbiota. We and others have shown that gut microbial mice with this cocktail for 7 days dramatically increased the level dysbiosis contributes to susceptibility toward colitis and CAC of Akkermansia, but decreased Prevotella to a level similar to that (36–38). IgA is known to regulate the gut microbial landscape, seen with metronidazole treatment (Supplemental Figure 7A). and mice deficient in IgA and pIgR are known to harbor a dys- Under this microbial landscape, colon explants from WT mice also biotic microbiota that contributes to their increased suscepti- released increased levels of IL-1α during DSS administration (Sup- bility to colitis (33, 39–41). We therefore posited that decreased plemental Figure 7B), and the treated mice displayed increased colonic IgA in Il33–/– mice would lead them to developing a dys- susceptibility to DSS-induced colitis (Supplemental Figure 7, C–E). biotic microbiota. To assess whether IL-33 has an effect on gut Therefore, overrepresentation of the mucolytic bacterium Akker- microbial homeostasis, we performed real-time quantitative mansia in the Il33–/– mice led to increased IL-1α release and ensu- PCR (qPCR) analysis of the gut microbiota derived from colon ing colitis after DSS administration. contents of separately housed WT and Il33–/– mice. Relative to IL-33 maintains gut homeostasis by modulating the IgA- WT mice, Il33–/– mice harbored increased levels of Akkermansia microbiota axes. Mice are coprophagus in nature, allowing for muciniphila and segmented filamentous bacteria (SFB), while the equilibration of gut contents by cohousing. Upon cohousing for levels of bacteria belonging to other class and phyla were similar 2 weeks, levels of Akkermansia equilibrated between cohoused (Figure 6, A and B). A. muciniphila is an anaerobic bacterium that WT and cohoused Il33–/– mice (Figure 7A), which correlated with specializes in degrading the mucus (42) and is highly targeted an increase in levels of IgA in the stool samples of the cohoused by intestinal IgA in healthy individuals (39, 41). Recent reports Il33–/– mice (Figure 7B). In line with these observations, the have shown that exogenous Akkermansia administration leads to cohoused Il33–/– mice were significantly protected from DSS- increased permeability of the mucus barrier and epithelial injury induced body weight loss, systemic wasting, and colon short- in mice after challenge with noxious agents (43, 44). ening (Figure 7, C–E). Histological analysis confirmed amelio- We therefore determined whether increased levels of Akker- ration of inflammation and epithelial hyperplasia in the colons mansia promote early IL-1α release and susceptibility of Il33–/– of the cohoused Il33–/– mice when compared with the separately mice to DSS-induced colitis. Metronidazole is an antibiotic spe- housed Il33–/– mice (Figure 7, F and G). cific to obligate anaerobes (45), and treating Il33–/– mice with To determine whether IL-33 actively regulates microbial metronidazole (2.5 mg/ml in 1% sucrose) for 5 days led to a sig- homeostasis, we cohoused WT and Il33–/– mice for 2 weeks and nificant decrease in the level of Akkermansia (Figure 6C). This then separated them for another 4 weeks. While cohousing equili- depletion of Akkermansia by metronidazole treatment was inde- brated the level of Akkermansia and intestinal IgA in the stool pendent of modulation of intestinal IgA level in Il33–/– mice (Figure samples, Il33–/– mice regained the separately housed gut ecologi- 6D), but still led to decreased IL-1α release from the colon during cal landscape of increased Akkermansia (Figure 7A) and decreased DSS administration (Figure 6E) and protected the Il33–/– mice from intestinal IgA levels (Figure 7B) after 4 weeks of separation. In

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Figure 7. IL-33 actively regulates intestinal IgA levels, microbial homeostasis, and susceptibility to colitis. (A) qPCR analysis of indicated bacteria from stool samples of separately housed (SH), cohoused (CH), or cohoused and then separated (CH and separate) WT and Il33–/– mice and (B) IgA measurement in stool samples by ELISA. (C) Body weight change during DSS administration. (D) Colon length measurements and (E) representative colon images at day 8 after DSS administration. (F) Colon histological analysis and (G) representative H&E-stained colon sections at day 8 after DSS administration. Original magnification, ×10. Data are representative of 2 independent experiments and were analyzed by Kruskal-Wallis test (A, B, D, and F) or 2-way ANOVA (C), followed by Holm-Šídák post test. Error bars represent mean ± SEM, and each symbol represents an individual mouse. n = 9–11 mice per group. ***P < 0.001; ****P < 0.0001.

line with these observations, DSS administration led to increased Discussion susceptibility toward DSS-induced colitis in the separated Il33–/– Only 25% of patients achieve sustained disease remission with mice (Figure 7, C–G). These data suggest that transfer of intesti- the currently available anti-TNF biologics, while another 30% nal IgA in the Il33–/– mice upon cohousing with WT mice actively require total colectomy (50). Therefore, further investigations regulates the load of mucolytic bacteria and confers protection into the cytokine networks that underlie IBD are required to from DSS-induced colitis. develop novel therapeutic strategies. Expression of IL-1α, IL-33, Finally, we used littermate controls to confirm that IL-33 regulates ST2, and sST2 are increased in the mucosa of IBD patients, and intestinal IgA production and colitis susceptibility independently of SNPs in IL33 and ST2 are associated with the development of IBD the genetic background and starting microbial landscape. Il33+/– mice (18–22). Other studies that utilized distinct models of colitis or had WT levels of intestinal IgA and Akkermansia load in the stool, exogenous IL-33 infusion have attributed the functions of upreg- demonstrating that Il33 is haplosufficient for these functions (Supple- ulating the production of mucus, type 2 cytokines and cells, and mental Figure 8, A and B). In line with these observations, Il33+/– mice Tregs to the IL-33/ST2 signaling pathway (24, 29, 30). Consistent were similar to WT controls in susceptibility to DSS-induced colitis with these studies demonstrating a protective role of IL-33 in the (Supplemental Figure 8, C–F). On the other hand, Il33–/– littermates gut, we show here that Il33–/– mice were more susceptible to DSS- that were separated from WT and Il33+/– littermates at weaning for induced colitis. However, Il33–/– mice did not have a defect in the 4 weeks harbored decreased levels of IgA and increased levels of expression of mucins, amphiregulin, type 2 cytokine or cellular Akkermansia in the stool samples (Supplemental Figure 8, A and B). response, or in the number of Tregs in the colon under basal con- In line with these observations, Il33–/– littermates displayed increased ditions or early during DSS administration. Instead, Il33–/– colon susceptibility to DSS-induced colitis (Supplemental Figure 8, C–F). explants released high levels of IL-1α early during disease onset. Therefore, IL-33 confers protection from colitis by regulating colonic Increased IL-1α was pathogenic, as its neutralization or genetic IgA production and inhibiting microbial dysbiosis. deletion markedly protected Il33–/– mice from colitis.

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IBD is a strong predisposing factor for CAC, and expression Knock-out Mouse Project (KOMP; https://www.komp.org/) and of IL33 and ST2 is increased in low-grade tumors, but decreased rederived in C57BL/6 mice in the colony. Mice were maintained in a in high-grade tumors, suggesting a protective role of IL-33 in CAC specific pathogen–free facility. (22). In line with this hypothesis, we showed that Il33-deficient Induction of colitis and tumors. For DSS models of colitis, mice were mice were highly susceptible to AOM/DSS-induced CAC. Further- administered the indicated dose of DSS (molecular weight 36-40 kDa; more, we showed that IL-1α was a major driver of colitis and CAC Affymetrix) for 6 days, followed by regular water. For neutralization of in both Il33-deficient and Il33-sufficient settings. Maywald et al. IL-1α, mice were injected with 100 μg of anti–IL-1α antibody (clone ALF- showed that IL-33 signaling promotes small intestinal tumorigen- 161, Bio X Cell) in 100 μl sterile PBS on days 1, 3, and 5 via retro-orbital esis in a model that is dependent on aberrant Wnt signaling instead route during DSS administration. The AOM/DSS model for colorectal of inflammation (51). Therefore, the role of IL-33 in restraining tumorigenesis has been described previously (61). For metronidazole colitis-associated colon cancer is due to its dominant role in pretreatment, mice were administered with 2.5 mg/ml metronidazole venting IL-1α–mediated overt inflammation. IL-1α–blocking mono- (Teva Pharmaceuticals) with 1% sucrose in drinking water. For cohous- clonal antibody was well tolerated in phase I clinical trials for pso- ing experiments, equal numbers of female WT and female Il33–/– mice riasis and solid tumors (52, 53). Therefore, further trials in testing were housed in the same cage for 2 weeks before DSS administration its efficacy in ameliorating IBD and CAC are warranted. and remained cohoused for the duration of the experiment. For separa- Immune system components can regulate the host microbiota tion after cohousing experiments, mice were cohoused for 2 weeks, fol- that modulate the susceptibility to colitis and CAC (38, 48, 54, 55). lowed by separation for 4 weeks before DSS administration. Intestinal IgA is one such immune component that preferentially Histology and microscopy analysis. Colon tissues were fixed in 10% targets colitogenic members of the microbiota, including SFB and formalin, embedded in paraffin, sectioned, and stained with H&E. His- A. muciniphila (34, 39, 41, 56, 57). Reciprocally, certain pathobi- tological analysis for inflammation, epithelial hyperplasia, and tumori- onts can produce proteolytic enzymes that degrade intestinal IgA genesis was performed by a board-certified pathologist (P. Vogel) as and thereby enhance colitis susceptibility (40). We showed here described previously (62). FISH for Akkermansia was performed by uti- that IL-33 was essential for efficient production of IgA in the intes- lizing Alexa Fluor 647–conjugated probe as described previously (63). tine, even under homoeostatic conditions. Similar to the role of Cytokines and immunoglobulin measurement. Cytokines in colon IL-33 in Treg differentiation (30), IL-33 promoted IgA production explants, homogenates, and sera were measured by ELISA. Culture of in a TGF-β–dependent manner. Consistent with decreased lev- colon explants (64) and protein extraction from frozen colon tissues els of intestinal IgA in Il33–/– mice, they also harbored a dysbiotic (62) was described previously. Briefly, for explant culture, 0.5 cm of microbiota associated with increased levels of SFB and, in particu- the distal colon was incubated in IMDM media supplemented with lar, A. muciniphila. Verrucomicrobia family member Akkerman- 10%FBS and penicillin, streptomycin, and gentamycin for 48 hours. sia expresses mucinases that allow it to degrade the inner mucus Cytokine levels are presented as pg or ng/ml of the clarified super- layer and utilize it as a carbon and nitrogen source (42). As a con- natant media. Explants were also weighed before incubation, and sequence, experimental Akkermansia colonization weakens the equivalent data are obtained if the cytokine levels are normalized to mucus barrier and promotes epithelial injury after administration their wet weight. The IL-18 ELISA was from eBioscience; multiplex of the cytotoxic agent heme (43). Repeated dosing with Akkerman- ELISAs were used for all other cytokines (Millipore) according to the sia also leads to increased severity of DSS-induced colitis in WT manufacturers’ instructions. Immunoglobulin ELISA was performed mice (44) and promotes colon tumorigenesis in a mouse model of by the C57BL/6-HRP clonotyping system (SouthernBiotech) per the aberrant Wnt signaling in the epithelium (58). In line with these manufacturer’s instructions. studies, we found that depletion of Akkermansia by metronidazole Real-time qPCR. RNA was isolated using the RNeasy Kit (Sigma- treatment protected Il33-deficient mice from DSS-induced colitis Aldrich) per the manufacturer’s instructions and converted into cDNA and associated epithelial hyperplasia. as described earlier (64). Gene expression was assessed using 2× It is of particular clinical relevance that Akkermansia has SYBR Green Master Mix according to the manufacturer’s instructions also been shown to improve obesity and metabolic disorders (Applied Biosystems). Sequences for qRT-PCR primers are listed in (59). We have also previously shown that the antibiotic cock- Supplemental Table 1. qPCR data were analyzed by the 2–ΔΔCT method, tail that increases the levels of Akkermansia also ameliorates with β-actin as the housekeeping gene. For gut microbiota charac- IL-1β–mediated autoinflammation (49). We showed here that terization, genomic DNA from feces was extracted using the QiAMP the same antibiotic cocktail also strikingly increased colitis DNA Stool Mini Kit (QIAGEN) with bead beating in tissue lyser (Qia- severity. Further, it was recently shown that oral delivery of gen) and used for real-time PCR amplification of indicated micro- IgA reactive to pathobionts can decrease colitis susceptibility biota components by SYBR Green Master Mix (Applied Biosystems) (39). Therefore, along with modulation of IL-1α and IL-33 lev- as described earlier (65). Data were analyzed with the 2–ΔΔCT method els, discovering tools that lead to specific manipulation of the using universal eubacteria primers as control. microbiota, such as a tailored diet, targeted antibiotic regimen, B cell class switching assay. The rbc-depleted splenocytes or sort- or oral IgA supplementation, holds great promise of ameliorat- purified CD19+ B cells from naive C57BL/6 WT mice were cultured in ing inflammatory diseases and cancer. media supplemented with anti-CD40 (1 ng/ml), LPS (1 μg/ml, Invivo- gen), TGF-β (1 ng/ml, R&D Systems), and/or IL-33 (30 ng/ml, Pepro- Methods tech) in the indicated combinations. After 4 days in culture, superna- Mice. C57BL/6 WT and Il1a–/– (60) mice were described previous- tant media were analyzed for immunoglobulins by the C57BL/6-HRP ly. Il33tm1(KOMP)Vlcg (Il33-deficient) embryos were obtained from the Clonotyping System (SouthernBiotech) per the manufacturer’s instruc-

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tions. Supernatant concentrations were 1:10,000 for IgM and 1:100 DS, RK, and PV. AM, DS, QZ, PV, and TDK performed formal for IgA and IgGs. Only absorbance values more than 3 SD away from analysis. AM wrote the original draft. AM, DS, and TDK reviewed the mean of negative control were considered positive. Cells were and edited the manuscript. Funding acquisition was by TDK. TDK used for intracellular staining of IgA (clone mA-E61, eBioscience) and provided resources. TDK supervised the project. analyzed by flow cytometry. Statistics. Body weight change data were analyzed by 2-way Acknowledgments ANOVA followed by the Holm-Šídák post test. Survival data were This work was supported by the NIH (AI101935, AI124346, analyzed by log-rank (Mantel-Cox) test. Statistical significance for AR056296 and CA163507 to TDK) and the American Lebanese other data sets was determined by parametric or nonparametric tests, Syrian Associated Charities (to TDK). We would like to thank Si where appropriate, and are indicated in the figure legends; P < 0.05 Ming Man and Prajwal Gurung for helpful discussions and editing was considered significant. of the manuscript. We would like to apologize to our colleagues Study approval. Animal study protocols were approved by the whose work could not be cited due to space limitations. St. Jude Children’s Research Hospital Committee on the Use and Care of Animals. Address correspondence to: Thirumala-Devi Kanneganti, Depart- ment of Immunology, St. Jude Children’s Research Hospital, MS Author contributions #351, 262 Danny Thomas Place, Memphis, Tennessee 38105-3678, AM and TDK conceptualized the project. Methodology was USA. Phone: 901.595.3634; E-mail: Thirumala-Devi.Kanneganti@ designed by AM, QZ, DS, and RK. Investigation was by AM, QZ, StJude.org.

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